Skip to main content
SpringerLink
Log in

Human AT1 receptor is a single copy gene: Characterization in a stable cell line

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

To address conflicting reports concerning the number of angiotensin II (AII) receptor type 1 (AT1) coding loci in vertebrates, Southern blot analysis was used to determine the genomic representation of AT1 receptor genes in animals comprising a divergent evolutionary spectrum. The data demonstrate that the AT1 receptor gene is present as a single genomic copy in a broad spectrum of animals including human, monkey, dog, cow, rabbit, and chicken. In contrast, members of the rodent taxonomic order contain two genes in their genomes. These two genes may have arisen in rodents as a consequence of a gene duplication event that occurred during evolution following the branching of rodents from the mammalian phylogenetic tree. In order to investigate the properties of the human AT1 receptor in a pure cell system, the recombinant human AT1 receptor was stably expressed in mouse L cells. An isolated cell line, designated LhAT1-D6, was found to express abundant levels of recombinant receptor [430±15 fmol/mg] exhibiting high affinity [KD=0.15±0.02 nM] for [125I][SAR1, IIe8] angiotensin II (SIA). The pharmacological profile of ligands competing for [125I] SIA binding to the expressed recèptor was in accordance with that of the natural receptor. Radioligand binding of the expressed receptor was decreased in the presence of the non-hydrolyzable analog of GTP, guanosine 5′-(γ-thio) triphosphate [GTPγS]. Angiotensin II evoked a rapid efflux of45Ca2+ from LhAT1-D6 cells that was blocked by AT1 receptor specific antagonists. In addition, AII inhibited forskolin-stimulated cAMP accumulation in these cells which was blocked by the AT-1 antagonist. Thus, the LhAT1-D6 cell line provides a powerful tool to explore the human AT1 receptor regulation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Peach MJ: Renin-angiotensin system: biochemistry and mechanisms of action. Physiol Rev 57: 313–370, 1977

    Google Scholar 

  2. Peach MJ: Molecular actions of angiotensin. Biochem Pharmacol 30: 2745–2751, 1981

    Google Scholar 

  3. Whitebread S, Mele M, Kamber B, de Gasparo M: Preliminary biochemical characterization of two angiotensin II receptor subtypes. Biochem Biophys Res Commun 163: 284–291, 1989

    Google Scholar 

  4. Chiu AT, Herblin WF, McCall DE, Ardecky RJ, Carini DJ, Duncia JV, Pease LJ, Wong PC, Wexler RR, Johnson AL, Timmermans PBMWN: Identification of angiotensin II receptor subtypes. Biochem Biophys Res Commun 165: 196–203, 1989

    Google Scholar 

  5. Timmermans PBMWM, Wong PC, Chiu AT, Herblin WF: Nonpeptide angiotensin II receptor antagonists. TIPS 12: 55–62, 1991

    Google Scholar 

  6. Hodges JC, Hamby JM, Blankey CJ: Angiotensin II receptor binding inhibitors. Drugs of the Future 17(7): 575–593, 1992

    Google Scholar 

  7. Wong PC, Chiu AT, Duncia JV, Herblin WF, Smith RD, Timmermans PBMWN: Angiotensin II receptor antagonists and receptor subtypes. Trends Endocrinol Metab 3: 211–217, 1992

    Google Scholar 

  8. Chiu AT, McCall DE, Price WA, Wong PC, Carini DJ, Duncia JV, Wexler RR, Yoo SE, Johnson AL, Timmermans PBMWM: Nonpeptide angiotensin II receptor antagonists. VII. Cellular and biochemical pharmacology of DUP 753, an orally active antihypertensive agent. J Pharmacol Exp Ther 252: 711–718, 1990

    Google Scholar 

  9. Weinstock J, Keenan RM, Samanen J, Hempel J, Finkelstein JA, Franz RG, Gaitanopoulos DE, Girard GR, Gleson JG, Hill DT, Morgan TM, Peishoff CE, Aiyar N, Brooks DP, Fredrickson TA, Ohlstein EH, Ruffolo RR Jr, Stack EJ, Sulpizio AC, Weidley EF, Edwards RM: 1-(carboxybenzyl) imidazole-5-acrylic acids: potent and selective angiotensin II receptor antagonists. J Med Chem 34: 1514–1517, 1991

    Google Scholar 

  10. deGasparo M, Whitebread S, Mele M, Motani AS, Whitcombe PJ, Ramjoue HP, Kamber B: Biochemical characterization of two angiotensin II receptor subtypes in the rat. J Cardiovas Pharmacol 16 (Suppl 4): S31-S35, 1990

    Google Scholar 

  11. Blankley CJ, Hodges JC, Klutchko SR, Himmelsback RJ, Chucholowski A, Connelly CJ, Neergaard SJ, Van Nieuwenhuize MS, Sebastian A, Quin J III, Essenburg AD, Cohen DM: Synthesis and structure-activity relationships of a novel series of nonpeptide angiotensin II receptor binding inhibitors specific for the AT2 subtype. J Med Chem 34: 3248–3260, 1991

    Google Scholar 

  12. Chiu AT, McCall DE, Nguyen TT, Carini DJ, Duncia JV, Herblin WF, Uyeda RT, Wong PC, Wexler RR, Johnson AL, Timmermans PBMWM: Discrimination of angiotensin II receptor subtypes by dithiothreitol. Eur J Pharmacol 170: 117–118, 1989

    Google Scholar 

  13. Dudley DT, Panek RL, Major TC, Lu GH, Bruns RF, Klinkefus BA, Hodges JC, Weishaar RE: Subclasses of angiotensir II binding sites and their functional significance. Mol Pharmacol 38: 370–377, 1990

    Google Scholar 

  14. Murphy TJ, Alexander RW, Griendling KK, Runge MS, Bernstein KE: Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor. Nature 351: 233–236, 1991

    Google Scholar 

  15. Sasaki K, Yamano Y, Bardhan S, Iwai N, Murray JJ, Hasegawa M, Matsuda Y, Inagami T: Cloning and expression of a complemen tary DNA encoding a bovine adrenal angiotensin II type-1 receptor. Nature 351: 230–232, 1991

    Google Scholar 

  16. Langford K, Frenzel K, Martin BM, Bernstein KE: The genomic organization of the rat AT1 angiotensin receptor. Biochem Biophys Res Commun 183: 1025–1032, 1992

    Google Scholar 

  17. Kakar SS, Sellers JC, Devor DC, Musgrove LC, Neill JD: Angiotensin II type-1 receptor subtype cDNAs: differential expression and hormonal regulation. Biochem Biophys Res Commun 183: 1090–1096, 1992

    Google Scholar 

  18. Flton TS, Stephan CC, Taylor GR, Kimball MG, Martin MM, Durand JN, Oparil S: isolation of two distinct type 1 angiotensin II receptor genes. Biochem Biophys Res Commun 184: 1067–1073, 1992

    Google Scholar 

  19. Ye MQ, Healy DP: Characterization of an angiotensin type-1 receptor partial cDNA from rat kidney: evidence for a novel AT1B receptor subtype. Biochem Biophys Res Commun 185: 204–210, 1992

    Google Scholar 

  20. Sasamura H, Hein L, Krieger JE, Pratt RE, Kobilka BK, Dzau VJ: Cloning, and characterization, and expression of two angiotensin receptor (AT-1) isoforms from the mouse genome. Biochem Biophys Res Commun 185: 253–259, 1992

    Google Scholar 

  21. Sandberg K, Ji H, Clark AJL, Shapira H, Catt KJ: Cloning and expression of a novel angiotensin II receptor subtype. J Biol Chem 267: 9455–9458, 1992

    Google Scholar 

  22. Bergsma DJ, Ellis C, Kumar C, Nuthulaganti P, kersten H, Elshourbagy N, Griffin E, Stadel JM, Aiyar N: Cloning and characterization of a human angiotensin II type 1 receptor. Biochem Biophys Res Commun 183: 989–995, 1992

    Google Scholar 

  23. Furuta H, Guo DF, Inagami T: Molecular cloning and sequencing of the gene encoding human angiotensin II type 1 receptor. Biochem Biophys Res Commun 183: 8–13, 1992

    Google Scholar 

  24. Takayanagi R, Ohnaka K, Sakai Y, Nakao R, Yanase T, Haji M, Inagami T, Furuta H, Gou D-F, Nakamuta M, Nawata H: Molecular cloning, sequence analysis and expression of a cDNA encoding human type-1 angiotensin II receptor. Biochem Biophys Res Commun 183: 910–916, 1992

    Google Scholar 

  25. Mauzy CA, Hwang O, Egloff AM, Wu L-H, Chung F-Z: Cloning, expression and characterization of a gene encoding the human angiotensin II type 1A receptor. Biochem Biophys res Commun 186: 277–284, 1992

    Google Scholar 

  26. Iwai N, Inagami T: Identification of two subtypes in the rat type 1 angiotensin II receptor. FEBS Lett 298: 257–260, 1992

    Google Scholar 

  27. Iwai N, Yamano Y, Chaki S, Konishi F, Bardhan S, Tibbetts C, Sasaki K, Hasegawa M, Matsuda Y, Inagami T: Rat angiotensin II receptor: cDNA sequence and regulation of the gene expression. Biochem Biophys Res Commun 177: 299–304, 1991

    Google Scholar 

  28. Kitami Y, Okura T, Marumoto K, Wakamiya R, Hiwada K: Differential gene expression and regulation of type-I angiotensin II receptor subtypes in the rat. Biochem Biophys Res Comm 188: 446–452, 1992

    Google Scholar 

  29. Saiki RK, Sharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N: Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350–1353, 1985

    Google Scholar 

  30. Sharf SJ, Horn GT, Erlich HA: Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science 233: 1076–1078, 1986

    Google Scholar 

  31. Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98: 503–517, 1975

    Google Scholar 

  32. connors RW, Sweet RW, Norveral JP, Pfarr DS, Trill JJ, Shebuski RJ, Berkowitz BA, Williams D, Franklin S, Reff ME: DHFR coamplification of t-PA in DHFR+ bovine endothelial cells:In vitro Characterization of the purified serine protease. DNA 7: 651–661, 1988

    Google Scholar 

  33. Aruffo A, Seed B: Molecular cloning of a CD28 cDNA by high-efficiency COS cell expression system. Proc Natl Acad Sci USA 84: 8573–8577, 1987

    Google Scholar 

  34. Pfarr DS, Sathe G, Reff ME: A highly molecular cloning vector for the analysis of eukaryotic genes and gene regulatory elements. DNA 4: 461–467, 1985

    Google Scholar 

  35. Southern PJ, Berg P: Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Gen 1: 327–341, 1982

    Google Scholar 

  36. Simonsen CC, Levinson AD: Isolation and expression of an altered mouse dihydrofolate reductase cDNA. Proc Natl Acad Sci USA 80: 2495–2499, 1983

    Google Scholar 

  37. Gluzman Y: SV40-transformed Simian cells supports the replication of early SV40 mutants. Cell 23: 175–182, 1981

    Google Scholar 

  38. Aiyar N, Nambi P, Stassen FL, Crooke ST: Vascular vasopressin receptors mediate phosphotidylinositol turnover and calcium efflux in an established smooth muscle cell line. Life Sci 39: 37–45, 1986

    Google Scholar 

  39. Gleason MM, Griffin EC, Nambi P, Aiyar N: Endothelin stimulates86Rb efflux in rat glioma C6-Bu-1 cells. Neuropeptides 20: 17–23, 1991

    Google Scholar 

  40. Quast U: Effect of the K+ efflux stimulating vasodilator BRL 34915 on86Rb+ efflux and spontaneous activity in guinea pig portal vein. Br J Pharmacol 91: 569–578, 1987

    Google Scholar 

  41. Holland SK, Blake CCF: Proteins, exons and molecular evolution. In: EM Stone and RJ Schwartz (eds.), Intervening Sequences in Evolution and Development, pp. 11–42, Oxford Univ Press, New York, NY, 1990

    Google Scholar 

  42. Murphy TJ, Nakamura Y, Takeuchi K, Alexander RW: A cloned angiotensin receptor isoform from the turkey adrenal gland is pharmacologically distinct from mammalian angiotensin receptors. Mol Pharmacol 44: 1–7, 1993

    Google Scholar 

  43. Yoshida H, Kakuchi JD, Guo DF, Furuta H, Iwai N, Van der Meerde-Jong R, Inagami T, Ichikawa I: Analysis of the evolution of angiotensin II type-1 receptor gene in mammals (mouse, rat, bovine and human): Biochem Biophys Res Commun 186: 1042–1049, 1992

    Google Scholar 

  44. Bergsma DJ, Ellis C, Nuthuluganti PR, Nambi P, Scaife K, Kumar C, Aiyar N: Isolation and expression of a novel angiotensin II receptor from Xenopus laevis heart. Mol Pharmacol 44: 277–284, 1993

    Google Scholar 

  45. Ji H, Sandberg K, Zhang Y, Catt KJ: Molecular cloning sequencing and functional expression of an amphibian angiotensin II receptor. Biochem Biophys res Commun 194: 756–762, 1993

    Google Scholar 

  46. Birnbaumer LJ, Abramowitz J, Brown AM: Receptor-effector coupling by G-proteins. Biochem Biophys Acta 1031: 163–224, 1990

    Google Scholar 

  47. Exton JH: Mechanism of action of calcium-mobilizing agonists: some variations on a young theme. FASEB J 2: 1668–1678, 1988

    Google Scholar 

  48. Ohnishi J, Ishido M, Shibata T, Inagami T, Murakami K, Miyazaki H: The rat angiotensin II AT1A receptor couples with three different signal transduction pathways. Biochem Biophys Res Commun 186: 1094–1101, 1992

    Google Scholar 

  49. Webb ML, Monshizadegan H, Dickinson KEJ, Serafino R, Moreland S, Michel I, Seiler SM, Murphy TJ: Binding and signal transduction of the cloned vascular angiotensin II (AT1A) receptor cDNA stably expressed in Chinese hamster ovary cells. regul Peptides 44: 131–139, 1993

    Google Scholar 

  50. Czelusniak J, Goodman M, Moncrief ND, Kehoe SM: Maximum parsimony approach to construction of evolutionary trees from aligned homologous sequences. Methods in Enzymol 183: 601–615, 1990.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, UW2510, P.O. Box 1539, 19406, King of Prussia, PA, USA

    Nambi Aiyar, Elayne Baker & Richard M. Edwards

  2. Department of Renal Pharmacology, SmithKline Beecham Pharmaceuticals, UW2510, P.O. Box 1539, 19406, King of Prussia, PA, USA

    Hsiao-Ling Wu & Ponnal Nambi

  3. Department of Gene Expression Sciences, SmithKline Beecham Pharmaceuticals, UW2510, P.O. Box 1539, 19406, King of Prussia, PA, USA

    John J. Trill

  4. Department of Molecular Genetics, SmithKline Beecham Pharamaceuticals, 19406, King of Prussia, PA, USA

    Catherine Ellis & Derk J. Bergsma

Authors
  1. Nambi Aiyar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elayne Baker
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hsiao-Ling Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ponnal Nambi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Richard M. Edwards
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. John J. Trill
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Catherine Ellis
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Derk J. Bergsma
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Aiyar, N., Baker, E., Wu, HL. et al. Human AT1 receptor is a single copy gene: Characterization in a stable cell line. Mol Cell Biochem 131, 75–86 (1994). https://doi.org/10.1007/BF01075727

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01075727

Key words

Search

Navigation